Effect of radiotherapy on head and neck cancer tissues in patients receiving radiotherapy: a bioinformatics analysis-based study

Radiotherapy is pivotal in treating head and neck cancers including nasopharyngeal, tongue, hypopharyngeal, larynx, maxillary sinus, parotid gland, and oral cancers. It holds the potential for curative effects and finds application in conjunction with chemotherapy, either as a radical method to preserve organ function or as an adjuvant postoperative treatment. We used bioinformatics analysis to investigate the effects of radiotherapy on head and neck cancer tissues in patients who had received radiotherapy. In this study, the expression and mutation profiles of The Cancer Genome Atlas–Head-Neck Squamous Cell Carcinoma were downloaded from the UCSC-Xena database, categorizing patients into two groups—those receiving radiotherapy and those not receiving radiotherapy. Subsequently, differential expression analysis and gene set enrichment analysis (GSEA) were performed. Following this, single-sample GSEA (ssGSEA) scores related to glucose and lipid metabolism were compared between the two groups. Additionally, immune cell infiltration analysis and single-cell verification were performed. Finally, the mutation profiles of the two groups were compared. The analyses revealed that patients receiving radiotherapy exhibited prolonged survival, enhanced apoptosis in head and neck cancer tissue, and diminished keratinocyte proliferation and migration. A comparison of ssGSEA scores related to glucose and lipid metabolism between the two groups indicated a reduction in glycolysis, tricarboxylic acid cycle activity, and fat synthesis in tissues treated with radiotherapy, suggesting that radiotherapy can effectively inhibit tumour cell energy metabolism. Analyses of immune cell infiltration and single-cell verification suggested decreased infiltration of immune cells post-radiotherapy in head and neck cancer tissues. A comparison of mutation profiles revealed a higher frequency of TP53, TTN, and CDKN2A mutations in patients receiving radiotherapy for head and neck cancer. In conclusion, the bioinformatics analyses delved into the effect of radiotherapy on patients with head and neck carcinoma. This study provides a theoretical framework elucidating the molecular mechanisms underlying radiotherapy's efficacy in treating head and neck cancer and presents scientific recommendations for drug therapy following radiotherapy.

Therapeutic approaches for HNC encompass minimally invasive, organ-sparing surgical techniques, advancements in radiotherapy, and curative multimodal strategies 9 .For patients with early-stage HNC, both surgery and intensive radiotherapy yield comparable outcomes concerning local disease control and overall survival.Following surgical intervention, postoperative radiotherapy, with or without adjunctive chemotherapy, is recommended for patients displaying pathological risk factors including perineural invasion, lymph vascular invasion, and positive or narrowly clear surgical margins [10][11][12] .Radiotherapy plays a pivotal role in the comprehensive treatment of HNC 10 .Chemoradiotherapy, initially used for managing inoperable disease, has now extended to postoperative treatment for high-risk patients, while its effectiveness in preserving organs is currently being assessed 13 .Simultaneously, radiotherapy can be used in combination with chemotherapy, serving as a radical approach to preserve organ function or as an adjuvant treatment after surgery 14 .
This study performed a comprehensive analysis involving differential expression assessment and gene set enrichment analysis (GSEA) utilizing The Cancer Genome Atlas-Head-Neck Squamous Cell Carcinoma (TCGA-HNSC) expression and mutant profiles from the UCSC-Xena database.The primary focus was to compare the single-sample GSEA (ssGSEA) scores related to glucose and lipid metabolism between distinct groups within this dataset.Additionally, immune cell infiltration analysis and assessments at the single-cell level were also performed.These analyses revealed significant alterations in tumour cell apoptosis, metabolism, immune microenvironment, and mutation profiles in patients with HNC following radiotherapy.These findings provide a theoretical basis for the molecular mechanisms underlying radiotherapy for HNC and offer crucial insights for devising effective drug therapies subsequent to radiotherapy.

Analysis of DEGs in head and neck cancer tissue treated with or without radiotherapy
Initially, according to clinical data from UCSC-Xena databases, patients from the TCGA-HNS cohort were segregated into those receiving radiotherapy and those not receiving.A comparative analysis of survival times was then performed between these groups using the log-rank test.The results indicated that patients who received radiotherapy exhibited significantly prolonged survival times and a more favorable prognosis (P < 0.0001; Fig. 1A).This underscores the efficacy of radiotherapy in extending survival among patients with HNC.Subsequently, to investigate alterations in the gene expression patterns of HNC tissue post-radiotherapy, DEGs between these groups were identified using the "limma" package in R, with criteria set at |log2FC|> 0.2 and P < 0.05 (Fig. 1B).Our analysis revealed that upregulated genes were associated with signaling pathways, apoptotic processes, and both positive and negative regulation of cell proliferation and migration, as well as physiological rhythm (Fig. 1C).Conversely, genes with downregulated expression were enriched in functions related to muscle contraction, cell adhesion, keratinization, epidermal development, angiogenesis, leukocyte migration, and keratinocyte proliferation (Fig. 1D).These findings suggest an improvement in the degree of apoptosis in the HNC tissues after radiotherapy, accompanied by reduced proliferation of keratinocytes, angiogenesis, and immune cell infiltration.
Genes with upregulated expression levels involved in the apoptotic process after radiotherapy were delineated (Fig. 1E).Notably, PPP1R15A emerged as a significant factor in mammalian integrative stress.PPP1R15A inhibitor, Sephin1, has shown potential in inhibiting the clonal expansion of tumour-specific T cells, thus posing as a promising target for tumour immunotherapy.Sephin1 also exhibits potential in treating immune-related diseases, including autoimmune conditions 15,16 .NFKBIA, a crucial regulator of NF-κB, has implications in glioma proliferation and drug resistance.Haploid deletions of NFKBIA exhibit distinct recurrence patterns in conjunction with other genetic markers and are disproportionately present upon recurrence, leading to adverse patient outcomes independent of genetic and clinicopathological predictors 17,18 .

GSEA validation
GSEA was performed to validate the enhanced degree of apoptosis and reduction in keratinocyte migration and proliferation in HNC tissues after radiotherapy.The analysis further revealed significant enrichment in biological processes in the radiotherapy group, including positive regulation of intrinsic apoptosis signaling pathways, endoplasmic reticulum stress-induced intrinsic apoptosis signaling pathways, and negative regulation of stem cell population maintenance (Fig. 2A).By contrast, the nonradiotherapy group exhibited significant enrichment in keratinocyte migration, positive regulation of glycolysis, and myoblast proliferation (Fig. 2B).

Metabolic reprogramming in head and neck cancer tissues treated with radiotherapy
Based on the GSEA findings, the nonradiotherapy group displayed a significant enrichment in glycolysis.This observation implies that radiotherapy has the potential to substantially reduce the ability of tumour cells for engaging in glycolysis, which serves as a primary mechanism for generating energy within these cells.Consequently, radiotherapy might hinder the proliferation and replication of tumour cells by inhibiting glycolysis.
To delve deeper into the alterations in glucose and lipid metabolism in HNC tissues after radiotherapy, genes associated with glucose and lipid metabolism pathways were retrieved from KEGG.Subsequently, the enrichment scores of these pathways in each sample were computed using ssGSEA.The higher the score, the stronger the activity of this pathway in the sample.Remarkably, significantly lower scores were observed for tricarboxylic acid (TCA) cycling, glycolysis/gluconeogenesis, starch, and sucrose metabolism in the radiotherapy group than in the nonradiotherapy group (Fig. 3A).Regarding lipid metabolism, the scores for fatty acid biosynthesis and sphingolipid metabolism in the radiotherapy group were also significantly lower than those in the nonradiotherapy group (Fig. 3B).These findings indicate that radiotherapy effectively diminishes the ability to carry out glycolysis, TCA cycle, and fat synthesis of tumour cells, thereby reducing the energy supply to tumour cells.www.nature.com/scientificreports/

Alteration of the immune microenvironment in head and neck cancer tissues after radiotherapy
The biological process of gene enrichment exhibiting down-regulated expression in the radiotherapy group involves leukocyte migration, indicating a potential weakening of immune resistance in patients with HNC after radiotherapy.To further investigate changes in the proportion of different immune cell infiltration postradiotherapy, information on 28 immune cell types and their corresponding marker genes were obtained [PMID:24138885].Subsequently, using the ssGSEA method, the enrichment scores of these immune cells in each HNC tissue we assessed using ssGSEA.Significantly lower scores were observed for central memory CD8 T cells, type 2 T helper cells, and neutrophils in the radiotherapy group than in the nonradiotherapy group (Fig. 4A).Furthermore, utilizing the CIBERSORT method, the proportion of infiltration of 22 immune cell types was calculated; the results revealed that the proportion of M1 macrophages, resting mast cells, and activated NK cells in the radiotherapy group was notably lower than that in the nonradiotherapy group.Conversely, the proportion of regulatory T cells was significantly higher in the radiotherapy group than in the nonradiotherapy group (Fig. 4B).

Validation of single-cell level
To further investigate shifts in the distribution of distinct cell types within HNC tissues following radiotherapy, the dataset GSE181919 was downloaded from the GEO database.After isolating cancer samples and employing cell filtration, SCT standardization, and dimension reduction clustering based on the official Seurat methodology, 9 primary cell types were identified, encompassing 23,088 cells (Fig. 5A).Subsequently, marker genes were used to annotate these cell types (Fig. 5B).Using DEGs for each cell type, the enrichment scores of individual cell types within HNC samples were computed using ssGSEA.Remarkably, significantly lower scores were observed for epithelial cells, NK/T cells, and plasma B cells in the radiotherapy group than in the nonradiotherapy group (Fig. 5C).These findings suggest a marked reduction in the proportion of tumour cells, as well as NK/T cells and plasma B cells, in HNC tissues treated with radiotherapy.

Alteration mutation profiles in head and neck cancers after radiotherapy
Given the potential DNA damage resulting from radiation exposure and its effect on genetic mutations, this study investigated changes in gene mutation patterns in HNC tissues following radiotherapy.Utilizing HNC mutation data sourced from the TCGA official website, patients were categorized into two groups: those receiving radiotherapy and those not receiving radiotherapy.Subsequently, the "oncoplot" function from the maftools package was utilized to visualize the top 20 genes with mutation frequencies in both groups.Noteworthy observations from the analysis revealed a significant increase in the mutation frequency of TP53 from 66 to 72% among patients with HNC after radiotherapy.Moreover, the mutation frequency of TTN surged from 29 to 39%, while CDKN2A increased from 18 to 22%.These findings strongly imply a potential association between radiotherapy and heightened mutation rates in TP53, TTN, and CDKN2A genes among patients with HNC (Fig. 6).Considering these results, it is recommended that after radiotherapy, patients with HNC might benefit from targeted drug therapies focusing on TP53, TTN, and CDKN2A.Examples of such drugs include olaparib for TP53 and Piperoxilil for CDKN2A.

Discussion
HNC represents a group of malignancies characterized by genetic complexity, posing significant challenges in management 19 .Annually, more than 880,000 new cases of HNC are diagnosed, leading to more than 450,000 fatalities 20 .These malignancies significantly impair patients' quality of life due to their poor prognosis, limited treatment responsiveness, and development of drug resistance 21 .A major hurdle in HNC treatment is the high recurrence and/or propensity to spread to other parts of the body, underscoring the intricate molecular landscape of this disease 22 .Despite the absence of specific targeted therapies, radiotherapy remains the primary treatment for HNC and is particularly advantageous for organ preservation 23 .It serves as a curative and palliative intervention across all disease stages.Technological advancements have notably enhanced the precision of radiotherapy, targeting tumours more accurately while minimizing radiation exposure to surrounding healthy tissues 24 .However, the effectiveness of radiotherapy is constrained by tumour hypoxia, which signifies an aggressive phenotype associated with increased metastasis and recurrence rates 25 .
In this regard, using clinical data obtained from the UCSC-Xena and TCGA-HNSC databases, this study categorized patients into cohorts: those receiving radiotherapy and those not receiving radiotherapy.A comparison of the survival duration of these cohorts using the log-rank test revealed that patients receiving radiotherapy exhibited prolonged overall survival and improved survival outcomes.This study conducted a detailed analysis of gene expression patterns in HNC tissues treated with radiotherapy to identify DEGs between the treated and untreated groups.Genes with upregulated expression were associated with several biological processes including signal transduction, apoptosis, and the regulation of cell proliferation, migration, and circadian rhythm.Conversely, genes with downregulated expression primarily contributed to muscle contraction, cell adhesion, keratinization, epidermal development, angiogenesis, leukocyte migration, and keratinocyte proliferation.Following radiotherapy, the expression of genes linked to apoptosis, such as KFKBIA, JTB, IGFBP3, BEX2, BBC3, and CXCR4, showed upregulation.A recent study demonstrated that IGFBP3, which is considered a potential biomarker for radiosensitivity, enhances cell death in oral squamous cell carcinoma cells during radiotherapy by inducing an increase in ROS through the activation of NF-κB and the production of cytokines 26 .BEX2, a gene expressed in the brain located on the X chromosome, is associated with cancer stem cells in cholangiocarcinoma.It is specifically expressed in dormant CSCs and plays a role in conferring resistance to chemotherapy 27 .Notably, the ubiquitin-binding enzyme E2C (UBE2C) was identified as playing a role in regulating oxidative stress and apoptosis-mediated resistance to radiotherapy in HNC.UBE2C was observed to reduce radiation-induced apoptosis by down-regulating oxidative stress and promoting the growth of malignant tumour cells 28 .Recent findings from both laboratory and patient-based research highlight that radiotherapy can effectively enhance anticancer properties by concurrently inhibiting immune checkpoint and angiogenesis pathways 29 .Targeted delivery of drugs to the CXCR4 presents a hopeful strategy for treating HNSCC.A recent investigation demonstrated that two types of nanotoxins equipped with T22 peptide ligands display potent CXCR4-dependent cytotoxic effects in vitro, and possess the ability to selectively target HNSCC cells that overexpress CXCR4.This represents a novel therapeutic approach for managing HNSCC 30 .
Glycolysis represents a biological pathway wherein glucose or glycogen breaks down into lactic acid, yielding a moderate production of ATP in the absence of sufficient oxygen 31 .Despite ample oxygen availability, cancer cells tend to produce energy through glycolysis, even in elevated oxygen levels 32 .Thus, inhibiting glycolysis alongside radiotherapy or chemotherapy has resulted in increased sensitivity and antitumour effects 33 .The results of ssGSEA revealed significantly lower scores for the TCA cycle, glycolysis/gluconeogenesis, starch, and sucrose metabolism in the radiotherapy group than in the nonradiotherapy group.Additionally, scores for fatty acid biosynthesis and sphingolipid metabolism were also significantly lower in the radiotherapy group than in the nonradiotherapy group.These findings suggest that radiotherapy effectively diminishes the rate of glycolysis, the TCA cycle, and fat synthesis in tumour cells, thereby reducing energy supply to tumour cells.Furthermore, the research unveiled that the biological mechanism of gene enrichment with decreased expression in the radiotherapy cohort pertains to the migration of white blood cells, indicating a compromised immune response in patients with HNC following radiotherapy.
Resistance to immunotherapy in HNC is associated with immune cell infiltration, tumour immunogenicity, and immune-related genes 34,35 .In the present study, immune cell infiltration analysis and single-cell level verification revealed a decrease in the proportion of immune cell infiltration in HNC tissues after radiotherapy.This suggests that patients who received radiotherapy should have an improved immunity.Lastly, when comparing the mutation profiles of the two groups, TP53, TTN, and CDKN2A mutations were more frequent in patients with HNC who received radiotherapy, suggesting that the importance of using combined drug therapy targeting TP53, TTN, and CDKN2A mutations in patients with HNC after radiotherapy.TP53 mutations are associated with the inactivation of normal p53 function or the acquisition of new functions that facilitate invasion, spread to other parts of the body, genetic instability, and cancer cell proliferation 36 .TTN mutations correlate with prognosis and increased tumour mutational burden in gastric cancer, indicating a negative prognosis in patients diagnosed with thyroid carcinoma 37,38 .Previous research has established a connection between CDKN2A and metabolic syndrome, hepatic fatty acid oxidation, ketogenesis, and adipose tissue differentiation 39 .A recent study has shown that the deletion of CDKN2A alters lipid metabolism in a way that enhances the susceptibility of glioblastoma to ferroptosis 40 .
While our research provides a theoretical framework for understanding the molecular mechanism of radiotherapy in treating HNC and offers guidance for post-radiotherapy drug therapy, there are several limitations www.nature.com/scientificreports/needing attention.The analysis in the present study is based on a public database; thus, further validation through clinical cohorts is necessary.Studies conducted in the future will aim to enhance the stability and reliability of the risk model through experimental validation and analysis of clinical samples to develop new, effective therapeutic strategies for prognosis assessment in HNC.In summary, this study focuses on metabolic changes in cell death, tumour cell genetic variation following radiotherapy in patients with HNC, and the immune system environment after radiotherapy.Bioinformatics analysis was used to investigate these effects and further confirmed our findings through single-cell analysis of alterations in various cell types within HNC tissues following radiotherapy.

Data source and preprocessing
The RNA-Seq and SNV data of TCGA-HNSC were retrieved from the UCSC-Xena databases and TCGA, respectively.GSE181919 was obtained from the Gene Expression Omnibus (GEO) databases, specifically selecting cells from cancer samples.This study employed Seurat's programming course, utilizing Read10X to read the data, preserving cells with gene detections ranging from 200 to 8000, and mitochondrial gene proportions below 10%.Subsequently, data normalization was performed using the SCTransform function.To address batch effects between samples, the harmony package was applied.Dimensionality reduction was executed using the RunUMAP function (dims = 1:20), followed by clustering of cell subpopulations utilizing the FindNeighbors and FindClusters functions (resolution = 0.1).The cell subgroups were categorized according to the marker genes of different cell types obtained from the CellMarker databases.

Analysis of DEGs
Detection of differentially expressed genes (DEGs) and comparison of the DEGs between the radiotherapy and nonradiotherapy groups were performing using the "limma" package in R, setting criteria of |log2FC|> 0.25 and p-value < 0.05.Genes with upregulated and downregulated expression were uploaded to the DAVID database to explore enriched biological processes (p < 0.05).

The Cancer Genome Atlas
The Cancer Genome Atlas (TCGA) serves to catalogue primary cancer-causing genomic alterations, contributing to the establishment of a comprehensive cancer genome atlas.These openly available datasets aid in improving diagnostic methods and treatment standards to prevent cancer 41 .The TCGA project encompasses more than 20 malignancy genomic datasets, providing crucial genetic and genomic insights into cancer 42 .

Analysis of GSEA
GSEA evaluates microarray data based on gene sets, interpreting gene expression data across diverse experimental methodologies such as RNA-seq, genome-wide association studies, proteomics, and metabolomics 43 .The strength of GSEA lies in providing a more stable and comprehensible measure of biological functions through gene sets 44 .It aids in analyzing and elucidating coordinated pathway-level changes in transcriptomics experiments.In this study, enriched biological processes were observed concerning the patient group receiving radiotherapy compared with that not receiving radiotherapy (P < 0.05).

Kyoto Encyclopedia of Genes and Genomes
Kyoto Encyclopedia of Genes and Genomes (KEGG) facilitates systematic analysis based on gene functions and connects genomic information with higher-order functional insights 45 .It is extensively utilized in transcriptomics, proteomics, glycomics, metabolomics, and metagenomics 46 .KEGG offers genome maps using Java graphics tools and serves as a computational tool for sequence comparison and path computation 45 .

Gene Expression Omnibus
The GEO (http:// www.ncbi.nlm.nih.gov/ geo/) serves as a public repository hosting diverse data sets from highthroughput gene expression and genomic hybridization experiments, encompassing platforms, samples, and series 47,48 .

Statistical analysis
Statistical analysis was performing by comparing continuous variables between the two groups using the Wilcoxon rank-sum test, setting statistical significance at P < 0.05.All analysis and image generation were executed using the R language (version 4.3.1).

Figure 1 .
Figure 1.Differential expression analysis of head and neck cancer tissues before and after radiotherapy.(A) Survival curve determined from The Cancer Genome Atlas-Head-Neck Squamous Cell Carcinoma in patients receiving or not receiving radiotherapy.(B) Volcano map of differentially expressed genes between groups with and without radiotherapy.(C) Biological process of the enrichment of genes with upregulated expression after radiotherapy.(D) Biological process enrichment of genes with downregulated expression after radiotherapy.(E) Apoptosis-related genes with upregulated expression after radiotherapy.

Figure 2 .
Figure 2. Gene set enrichment analysis (GSEA) validation.(A) Biological processes enriched in the patient group receiving radiotherapy in GSEA.(B) Biological processes enriched in the patient group not receiving radiotherapy in GSEA.

Figure 3 .
Figure 3. Metabolic reprogramming of head and neck cancer tissues after radiotherapy.(A) Single-sample gene set enrichment analysis (ssGSEA) scores of glucose metabolism between the radiotherapy and nonradiotherapy groups; (B) ssGSEA scores of lipid metabolism between the radiotherapy and nonradiotherapy groups.

Figure 4 .
Figure 4. Changes in the immune microenvironment in head and neck cancer tissues after radiotherapy.(A) Enrichment scores of 28 kinds of immune cells between the two groups were calculated using single-sample gene set enrichment analysis.(B) Proportion of 22 kinds of immune cells in the two groups was calculated using CIBERSORT.

Figure 5 .
Figure 5. Verification at the single-cell level.(A) UMAP of single-cell clusters and annotations in the GSE181919 cancer tissue sample.(B) Marker genes used to annotate cell types.(C) Single-sample gene set enrichment analysis scores for each cell type in the group receiving and not receiving radiotherapy.

Figure 6 .
Figure 6.Changes in the mutation profile of head and neck cancer tissues after radiotherapy.(A) Mutation profile of head and neck cancer tissues in patients not receiving radiotherapy.(B) Mutation profile of head and neck cancer tissues in patients receiving radiotherapy.